Advances in the study of molecules have been led, in part, by improvement in technologies used to characterise the molecules or their biological reactions. In particular, the study of nucleic acid, DNA and RNA, has benefitted from developing technologies used for sequence analysis and the study of hybridisation events.
An example of the technologies that have improved the study of nucleic acids, is the development of fabricated arrays of immobilised nucleic acids. These arrays typically consist of a high-density matrix of polynucleotides immobilised onto a solid support material. Fodor et al., Trends in Biotechnology (1994) 12:19-26, describes ways of assembling the nucleic acid arrays using a chemically sensitised glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotides.
An alternative approach is described by Schena et al., Science (1995) 270:467-470, where samples of DNA are positioned at predetermined sites on a glass microscope slide by robotic micropipetting techniques. The DNA is attached to the glass surface through its entire length by non-covalent electrostatic interactions.
The arrays are usually provided to study hybridisation events, determine the sequence of DNA (Mirzabekov, Trends in Biotechnology (1994) 12:27-32) or to detect mutations in a particular DNA sample. Many of these hybridisation events are detected using fluorescent labels attached to nucleotides with fluorescence detected using sensitive fluorescent detector, e.g. charge coupled detector (CCD). However, the major disadvantages of these methods are that it is not possible to sequence long stretches of DNA and repeat sequences can lead to ambiguity in the results. These problems are recognised in Automation Technologies for Genome Characterisation, Wiley—Interscience, 1997, Ed. T. J. Beugelsdijk, Chapter 10: 205-225.
In addition, the use of multi-molecule high-density arrays in a multi-step analysis procedure can lead to problems with phasing. Phasing problems result from a loss in the synchronisation of a reaction step occurring on different molecules of the array. If a proportion of the arrayed molecules fails to undergo a step in the procedure, subsequent results obtained for these molecules will no longer be in-step with results obtained for the other arrayed molecules. The proportion of molecules out of phase will increase through successive steps and consequently the results detected will become ambiguous. This problem is recognised in the sequencing procedure described in U.S. Pat. No. 5,302,509.